Use of avermectin derivative for increasing bioavailability and efficacy of macrocyclic lactones

ABSTRACT

The present invention relates to the use of avermectin derivative as a drug for the treatment of parasitic infections. The avermectin derivative is represented by the formula (I) wherein: (i) R 1  is chosen from the group constituted of —CH(CH 3 ) 2 , —CH(CH 3 )CH 2 CH 3 , or cyclohexyl, (ii) X represents —CH 2 —CH 2 —, or —CH═CH—, (iii) R 2  is chosen from 
                         
or an OH group, (iv) R 3  is OH or NOH, (v)   represents a single bond when R 3  is OH, or a double bond when R 3  is NOH, as an inhibitor of a membrane-bound protein which transports exogenous compounds out of target cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/352,669, filedApr. 17, 2014, which is the U.S. National Stage Entry ofPCT/EP2012/070704, filed Oct. 18, 2012, which claims priority toEuropean Patent Application No. 11306348.1, filed Oct. 18, 2011, theentire disclosures of each of which are incorporated herein byreference.

The present invention relates to the use of avermectin derivative inassociation with macrocyclic lactones as a formulation for the treatmentof parasitic infections.

Parasitic infections are the most frequent diseases for livestock ordomestic animal. In spite of recent advance in veterinary pharmaceuticalresearch, it is always necessary to find out more efficient and safedrug or formulation to fight against parasitic infections. For humans,the parasitic infections such as onchocerciasis caused by infection byOnchocerca volvulus, lymphatic filariasis caused by Wuchereriabancrofti, Brugia malayi, and Brugia timori, or tropical parasiticdiseases are still frequent diseases in developing countries. By theway, though endemic in some developing countries, intestinalstrongyloidiasis and cutaneous parasitic diseases also pose a threat tothe developed world.

The macrocyclic lactones (ML) are a family of broad spectrumanti-parasitic drugs that was developed in early 80s and have beenwidely used for the treatment of both internal and external parasites inpets, in livestock and in humans. MLs are also efficient for treatingparasitic diseases caused by benzimidazole-, levamisole-, andpyrantel-resistant strains of nematodes.

MLs are a family of compounds isolated from soil microorganismsbelonging to the genus Streptomyces. Macrocyclic lactones compriseavermectins and milbemycins. Avermectins comprise ivermectin, abamectin,doramectin, eprinomectin or selamectin, while milbemycins comprisemoxidectin, nemadectin, and milbemycin oxime.

The principal action of MLs in parasitic nematodes is to increasemembrane permeability to chloride ions by interacting with theglutamate-gated chloride channel subunit. The glutamate andγ-aminobutyric acid (GABA)-agonist activities of the MLs are themechanisms that lead to the paralysis and death of the treated parasitesat nanomolar concentration. In fact, MLs maintain open theglutamate-gated channels that blocks pharyngeal pumping and inhibits offeeding, which is one of the effects that cause the death of parasite.By the way, ivermectin leads also to activation and paralysis of bodymuscle in Haemonchus contortus (Sheriff et al., Vet. Parasitol., 2005,128(3-4), 341-346), and inhibits worm reproduction in Onchocercavolvulus (Schulz-Key, Acta Leiden, 1990, 59(1-2), 27-44).

However, in the last years, widespread MLs resistance has been observedin some nematode parasites of sheep, goats and cattle. The cause andmechanism of MLs resistance are yet not completely understood, butrecent research has showed that MDR (multidrug resistance) transportersare a group of protein implied in the MLs resistance. MDR transportersare membrane proteins belonging to the ABC (ATP binding cassette)family, and whose main function is the ATP-dependent transport of anumber of structurally unrelated exogenous compounds. Due to theirexpression in the plasma membrane, they function as a permeabilitybarrier for the passage of xenobiotic across the cell membrane byactively expelling them out of the cells. MDR transporters have beenconsidered as one of the causes of chemotherapy effectivenessrestriction, when the tumor cells overexpress these transporters. MDRtransporters also limit the entry of MLs into human target organism andaffect the efficiency of MLs as antiparasitic. In addition, theexpression of MDR transporters in intestine, liver and kidney allowsthem to detoxify these tissues, and ultimately eliminate the substratedrugs out of the systemic circulation, exerting a protecting actionagainst their toxicity but also restricting their therapeutic efficacy.

P-glycoprotein (Pgp), localized in the apical membrane, is one of MDRtransporters. The main function of Pgp is the active efflux of variousstructurally unrelated exogenous compounds to protect both vertebrateand invertebrate organisms against potentially toxic molecules. Pgp cantransport its substrate from the baso-lateral side to the apical side ofepithelia and endothelia. Pgp plays also an important role inblood-brain barrier, since it can limit the concentration of xenobioticsin the brain. The overexpression of Pgp is one of cause of drugresistance observed during avermectin treatment for parasitic infectionsor some tumor chemotherapy.

Later, other multidrug resistance proteins MRP1, 2 and 3 (ABCC1, 2 and3) are also discovered. They are also involved in multidrug resistanceand provide complementary and overlapping activities as multispecificdrug efflux pumps

The more recently discovered Breast Cancer Resistance protein is ABCG2,which assists Pgp to prevent unwanted material in the circulation frompassing into the brain. Homologues of MDR transporters exist also inparasite, and the selection and/or modulation of expression of theirgene could be one of the reasons of the resistance of parasite to MLs.

Different methods have been developed in the past years to overcome theeffectiveness restriction due to the efflux pumps of administrated drug.One of them is the use of MDR transporters inhibitors which can blockefflux pumps of administrated drugs to improve intracellularconcentration of active ingredient. Some Pgp ligands have been reportedin the past, such as cyclosporine A, its derivative PSC833 (Valspodar®),the antidiarrheal opioid drug loperamid, or verapamil. However,unfortunately, till today, because of important toxicity of these MDRtransporters inhibitors, neither of them can be applied inpharmaceutical use.

MLs have been firstly observed as substrates of MDR transporters. Later,it was found that MLs, in particular ivermectin, are also inhibitors ofMDR transporters.

In spite of the fact that ivermectin can efficiently inhibit MDRtransporters, it can not become a candidate drug, because of itsimportant neurotoxicity if it penetrates in the brain at highconcentration. In fact, ivermectin interacts with GABA receptors, acomplex situated in nervous system. The abnormal function of GABAreceptors can lead to neurologic, mental, vegetotropic, somatic,hormonal and other disorders. Since inhibition of MDR transporters isstill the most promising method to restrain chemotherapy resistance; andmacrocyclic lactone are still key molecules for treating parasiticinfections, it is necessary and urgent to find new safe and efficientinhibitors of MDR transporters.

The objective of the present invention is to provide an inhibitor ofmultidrug resistance proteins.

Particularly, the present invention concerns the use of an avermectinderivative compound of formula I

wherein:

(i) R₁ is chosen from the group constituted of —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, or cyclohexyl,

(ii) X represents —CH₂—CH₂—, or —CH═CH—,

(iii) R₂ is chosen from the group constituted of

or —OH group,(iv) R₃ is OH or NOH,(v)

represents a single bond when R₃ is OH, or a double bond when R₃ is NOH,as an inhibitor of a membrane-bound protein which transports exogenouscompounds out of target cells.

When R₂ represents —OH group, the compound of formula I is an aglyconeavermectin.

When R₂ represents

the compound of formula I is a monosaccharides of avermectin.

The Inventors of the present invention have surprisingly observed thataglycone avermectins or monosaccharide of avermectins expose acomparable inhibitory potency and efficiency with that of ivermectin orValspodar®, the last is one of the most efficient MDR inhibitors alreadyknown. Moreover, the Inventors have observed that aglycone avecmectinsor monosaccharide of avermectins have a higher inhibitory potency fornematode Pgp than that for murine Pgp. This particularity enablesaglycone avermectin or monosaccharide of avermectins to be used asadjuvant for conventional antiparasitic, which suffer from an efficiencyrestriction due to efflux pump by intermediate of Pgp of parasite. Themost surprisingly, aglycone avermectins or monosaccharide of avermectinsexhibit a weak agonist for GABA receptors, which means that aglyconeavermectins have weaker neurotoxicity compared to avermectin, especiallyivermectin.

“A membrane-bound protein which transports exogenous compounds out oftarget cells” can be a membrane-bound ATP-binding cassette (ABC)transporter protein which mediates cellular efflux of distinct drugs orchemicals of a wide variety of structure and function. Particularly,such membrane-bound protein can be P-glycoprotein (ABCB1), multidrugresistance associated protein family, including MRP1/ABCC1, MRP2, MRP2,or breast cancer resistant protein (ABCG2).

The inhibitor of such membrane-bound protein is a compound which canbind to said membrane-bound protein and thus reduce the affinity of saidmembrane-bound transporter with another substrate. The inhibitorypotency of an inhibitor can be measured according to any conventionalmethod, such as using a reference fluorescent substrate (ex: rhodamine123 for Pgp) of the transporter and by measuring the intracellularaccumulation of this substrate.

Another aspect of the present invention concerns the use of a compoundof formula I, as adjuvant for increasing bioavailability of an activeingredient of a drug whose efflux out of target cells depends on amembrane-bound protein which transports exogenous compounds out oftarget cells.

The term “adjuvant” refers to a molecule which has no therapeuticpotency when it is administrated alone, but can improve therapeuticpotency of another molecule when it is simultaneously administrated withsaid another molecule.

The avermectin derivative compound of the present invention, whichefficiently inhibits a membrane-bound protein, in particular ABCproteins, enables to improve intracellular concentration of the activeingredient of a drug, consequently, to restore or improve efficiency ofsaid drug.

More particularly, the present invention is related to avermectinderivative compounds of formula I:

wherein:(i) R₁ is chosen from the group constituted of —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, or cyclohexyl,(ii) X represents —CH₂—CH₂—, or —CH═CH—,(iii) R₂ is chosen from the group constituted of

or —OH group,(iv) R₃ is OH or NOH,(v)

represents a single bond when R₃ is OH, or a double bond when R₃ is NOH,for its use as an adjuvant of a drug.

In one particular embodiment, the invention concerns an avermectinderivative compound of formula I for its aforementioned use, wherein:

(i) R1 is chosen from the group constituted of —CH(CH₃)₂ and—CH(CH₃)CH₂CH₃,

(ii) X represents —CH₂—CH₂—,

(iii) R₂ is —OH,

(iv)

is —OH,

said compound corresponding to ivermectin aglycone of formula I(a):

In another particular embodiment, the invention concerns an avermectinderivative compound of formula I for its aforementioned use, wherein:

(i) R1 is chosen from the group constituted of —CH(CH₃)₂, and—CH(CH₃)CH₂CH₃,

(ii) X represents —CH₂—CH₂—,

(iii) R2 is

(iv)

R₃ is —OH,said compound corresponding to monosaccharide of ivermectin of formulaI(b).

In another particular embodiment, the invention concerns an avermectinderivative compound of formula I for its aforementioned use, wherein:

(i) R1 is chosen from the group constituted of —CH(CH₃)₂, and—CH(CH₃)CH₂CH₃,

(ii) X represents —CH═CH—,

(ii) R₂ is —OH,

(iv)

R₃ is —OH,

said compound corresponding to formula I(c):

A compound of formula I(c) can be eprinomectin aglycone, eprinomectinmonosaccharide, emamectine aglycone, emamectine monosaccharide,abamectine aglycone or abamectine monosaccharide.

In another particular embodiment, the invention concerns an avermectinderivative compound of formula I for its aforementioned use, wherein:

(i) R1 is cyclohexyl,

(ii) X represents —CH═CH—,

(iii) R₂ is —OH or

(iv)

R₃ is —OH,said compound corresponding to doramectin monosaccharide or doramectineaglycone of formula I(d):

In another particular embodiment, the invention concerns an avermectinderivative compound of formula I for its aforementioned use, wherein:

(i) R1 is cyclohexyl,

(ii) X represents —CH₂—CH₂—,

(iii) R₂ is —OH or

(iv)

is ═NOH,said compound corresponding to selamectin or selamectin aglycone offormula I(e):

In one particular embodiment, the avermectin derivative compound of thepresent invention is used as an adjuvant of an active ingredient chosenfrom the group comprising an antiparasitic, an antitumor agent, anantiviral agent, an anti-epileptic agent, an antibacterial agent, inparticular an antibiotic, an antifungal or any compound which issubstrate of said membrane-bound protein.

Such active ingredient can be any active ingredient used in anantiparasitic, antitumor agent, antiviral agent, or anti-epileptic agentknown in the art.

In one particular embodiment, the antiparasitic is chosen from the groupcomprising macrocylic lactones, such as the avermectins, in particularivermectin, abamectin, doramectin, eprinomectin or selamectin, or themilbemycins, in particular moxidectin, nemadectin, or milbemycin oxime.

In another particular embodiment, the antitumor agent is chosen from thegroup comprising:

-   -   antibiotic antitumor of type anthracycline, such as        daunorubicin, doxorubicin, mitocycin C, mitoxantron, adriamycin,        and actinomycin, or    -   taxanes, such as docetaxel, paclitaxel, or    -   alcaloides, such as vinblastin, vincristin, or    -   epipodophyllotoxins, such as etoposide, irinotecan, teniposide,        et topotecan.

In another particular embodiment, the antiviral agent is chosen from thegroup comprising: HIV-1 protease inhibitors, ritonavir, saquinavir,nelfinavir and indinavir and non-nucleoside reverse-transcriptaseinhibitors such as efavirenz.

In another particular embodiment, the anti-epileptic agent is chosenfrom the group comprising: Phenobarbital (PB;5-ethyl-5-phenyl-2,4,6-trioxohexahydropyrimidine), topiramate,lamotrigine phenytoin (PHT; 5,5-diphenyl-2,4-imidazolidinedione), andcarbamazepine (CBZ; 5H-dibenz[b,f]azepine-5-carboxamide).

In another particular embodiment, the antibacterial agent can be anantibiotic, such as loperamide, monensin, or the macrolides.

In another particular embodiment, the antifungal agent is chosen from anazole antifungal, such as itraconazole or ketoconazole.

Another aspect of the present invention is to provide a compositioncomprising a compound of formula I, in particular I(a), I(b), I(c), I(d)or I(e) for its use as drug.

Particularly, the present invention concerns a composition comprising acompound of formula I, in particular I(a), I(b), I(c), I(d) or I(e) forits use as drug in the treatment of parasite infections, viralinfections, chemotherapy resistant cancers, epilepsy, bacterialinfections or fungal infections.

The present invention concerns also a synergic composition comprising:

-   -   a compound of formula I, in particular I(a), I(b), I(c), I(d) or        I(e),    -   an active ingredient chosen from antiparasiticide, an antitumor        agent, an antiviral agent, an anti-epileptic agent, an        antibacterial agent, in particular an antibiotic, or an        antifungal agent.

More particularly, the composition of the present invention comprises:

-   -   a compound of formula I, in particular I(a), I(b), I(c), I(d) or        I(e), and    -   an active ingredient chosen from an antiparasiticide, an        antitumor agent, an antiviral agent, an anti-epileptic agent, an        antibacterial agent, in particular an antibiotic, or an        antifungal agent,        for its use as drug in the treatment of parasite infections,        viral infections, chemotherapy resistant cancers, epilepsy,        bacterial infections or fungal infections.

The present invention provides also a pharmaceutical compositioncomprising:

-   -   a compound of formula I, in particular I(a), I(b), I(c), I(d) or        I(e), and optionally    -   an active ingredient chosen from an antiparasiticide, an        antitumor agent, an antiviral agent, an anti-epileptic agent, an        antibacterial agent, in particular an antibiotic, or an        antifungal agent, and    -   a pharmaceutically acceptable carrier.

A pharmaceutically acceptable carrier can be any conventionalpharmaceutically acceptable carrier.

The pharmaceutical composition according to the present invention can beused in the treatment of parasite infections, viral infections,chemotherapy resistant cancers, epilepsy, bacterial infections or fungalinfections.

The pharmaceutical composition according to the present invention can beadministrated by oral route, subcutaneous injection, intravenousinjection, or intra-tissue injection.

The pharmaceutical composition according to the present invention can beadministrated with a diary dose from 0.01 mg/kg to 0.5 mg/kg

The present invention concerns also a kit which is a product containing

-   -   a compound of formula I, and    -   an active ingredient chosen from an antiparasitic, an antitumor        agent, an antiviral agent, an anti-epileptic agent, an        antibacterial agent, in particular an antibiotic, or an        antifungal agent,        as a combined preparation for simultaneous, separate or        sequential use in the treatment of parasite infections, viral        infections, chemotherapy resistant cancers, epilepsy, bacterial        infections or fungal infections.

The present invention is illustrated in detail by following figures andexamples. However, in any way, the figures and the examples can not beconsidered as a limitation of the scope of the present invention.

FIGURES

FIG. 1A represents the HPLC profile of ivermectin and ivermectinaglycone.

FIG. 1B represents the HPLC profile of ivermectin aglycone andmonosaccharide of ivermectin.

FIG. 2 represents the mass spectrometric profile of aglycone ivermectin.

FIG. 3 compares the maximum effective concentration of Valspodar® at 5μM (VSP, grey column), ivermectin at 5 μM (IVM, white column) andivermectin aglycone at 10 μM (Agly IVM, black column) in LLCPK1 cellstransfected with murine Pgp. Cells are incubated in a buffer containingrhodamine 123 with or without increasing concentrations of drugs andintracellular fluorescence was determined. Y axis representsintracellular fluorescence expressed as percent of the control value(cell incubated without drug). Look at also example 2.2.

FIG. 4A compares the inhibition of murine Pgp by ivermectin (opensquare) with that of ivermectin aglycone (black square) inLLC-PK1-mdr1a. X axis represents concentration of ivermectin orivermectin aglycone. Y axis represents intracellular rhodamineaccumulation compared to control value. Look at also example 2.3.

FIG. 4B compares the inhibition of nematode Pgp (HcPgpA) by ivermectin(open square) with that of ivermectin aglycone (black square) inLLC-PK1-HcPgpA. X axis represents concentration of ivermectin orivermectin aglycone. Y axis represents intracellular rhodamineaccumulation compared to control value. Look at also example 2.3.

FIG. 5 shows concentration-response curves of rat GABA(A) receptorexpressed in Xenopus oocytes. Concentration-dependent potentiation ofthe GABA receptor, presented as the percentage of the GABA-evokedresponse at EC₁₀ (2 μM). Y-axis represents normalized response to GABAreceptor according to the protocol described in the part 1.7 below.X-axis represents the concentration of moxidectin (MOX), ivermectin(IVM), ivermectin monosaccharide (IVM Monosaccharide), or ivermectinaglycone (IVM aglycone). Data were fitted to the Hill equation and aregiven as mean±S.D. Look at also example 2.4

FIG. 6 illustrates toxicity of ivermectin (open circle) and ivermectinaglycone (black circle) in Pgp-deficient mice. X axis represents thedose of ivermectin or ivermectin aglycone administrated to mice. Y axisrepresents the percentage of survival mice after one weekadministration. Look at also example 2.5.

FIG. 7 illustrates the reversion of drug-resistance by ivermectinaglycone in human lymphoma parental CEM cells and in vinblastineresistant CEM/VBL cells. CEM/VBL cells were incubated 4 days withvinblastine alone from 0 to 1 μg/ml (black square), or with vinblastinefrom 0 to 1 μg/ml and ivermectin (IVM) at 2.5 μM (open square), or withvinblastine from 0 to 1 μg/ml and ivermectine aglycone (IVM-Agly) at 2.5μM (open circle), or with vinblastine from 0 to 1 μg/ml and ivermectineaglycone (IVM-Agly) at 5 μM (black circle). CEM cells were incubated 4days with vinblastine alone from 0 to 1 μg/ml (—*—). X axis representsvinblastine (VBL) concentration. Y axis represents cytotoxicitydetermined using the MTT test. Values are mean±S.E.M. of 2 experiments(3 wells per experiment). Look at also example 2.6.

FIG. 8 illustrates the reversion of drug-resistance by ivermectinaglycone in multidrug resistant cells DC-3F/ADX which are resistant toactinomicyne D. Multidrug resistant cells DC-3F/ADX were incubated 3days with actinomycin alone from 0.01 to 10 μM (open square), or withactinomycin from 0.01 to 10 μM and ivermectin (IVM) at 5 μM (greysquare) or with actinomycin from 0.01 to 10 μM and ivermectin aglycone(IVM-Agly) at 5 μM (black triangle). X axis represents actinomycin Dconcentration. Y axis represents cytotoxicity determined using the MTTtest. Values are mean±S.E.M. of 2 experiments (3 wells per experiment).Look at also example 2.6.

FIG. 9 shows reversion of ivermectin-resistance by ivermectin aglyconein Caenorhabditis elegans resistant to ivermectin. Ivermectin resistancein C. elegans is determined according to the protocol described in part1.9 below. X axis represents ivermectin concentration. Y axis representsthe percentage of gravidity compared to control. Gravidity was evaluatedin the presence of ivermectine (IVM) alone at 0, 1, 2, 4, 6, 8, 10, 20ng/ml (open circle), or ivermectin at 0, 1, 2, 4, 6, 8, 10, 20 ng/mlwith verapamil at 8 μM (—x—), or ivermectin at 0, 1, 2, 4, 6, 8, 10, 20ng/ml with ivermectin aglycone (IVM Agly) at 10 ng/ml (11.4 nM) (blacksquare). Assays were performed in 3 replicates per condition treatmentand the experiment was performed 3 times. Mean±S.D. Look at also example2.7.

EXAMPLES

1. Materials and Methods

1.1 Ivermectin Aglycone Synthesis

Ivermectin aglycone (22,23-dihydroavermectin B1 aglycone) is obtainedfrom ivermectin by acid hydrolysis (1% of sulphuric acid). Ivermectinaglycone is purified by HPLC according to the method described byAlvinerie et al. (Ann Rech Vet, (1987), 18, 269-274).

1.2 Ivermectin Aglycone Structure Analysis

HPLC

The protocol of HPLC experiment is as follows: the product obtainedafter synthesis reaction is analysed by HPLC according a modified methodroutinely used in the INRA laboratory. Briefly a fluorescent derivativewas obtained by dissolving the eluent in N-methylimidazole andtrifluoroacetic anhydride (Aldrich, Milwaukee, Wis., USA) solutions inacetonitrile. The chromatographic conditions included a mobile phase ofacetic acid 2%, methanol, acetonitrile (4:32:64, v/v/v) pumped at a flowrate of 1.5 ml/min through a Supelcosil C18, 3 μm column (150×4.6 mm)(Supelco, Bellefonte, Pa., USA). Fluorescence detection (Detector RF551, Shimadu, Kyoto, Japan) was performed at 365 nm excitation and 475nm emission wavelength. The validation of the technique was performed(Alvinerie at al, 1993, Vet Res 24 (5): 417-21).

Mass Spectrometer

Structural characterization of the purified products was conducted onthe platform Axiom of INRA/ToxAlim, on a LCQ quadrupole ion trap massspectrometer (Thermo Finnigan, Les Ulis, France) fitted with anelectrospray ionization source operated in the positive mode. Theprotocol of mass spectrometer assay is as follows: collected sampleswere introduced into the ionization source by infusion at a flow rate of5 L/min with a syringe pump.

1.3 Cell Culture

The cells used were LLC-PK1, pig kidney epithelial cell lines, andLLC-PK1-mdr1a which are recombinant LLC-PK1 cells overexpressing murineabcb1a gene. All cell lines are available in INRA laboratory. Thetransfected cell line LLC-PK1-HcPgpA, which overexpress nematodeHaemonchus contortus PgpA, was developed by R. Prichard (McGillUniversity). Cells were cultured in medium 199 supplemented withpenicillin (100 units/ml), streptomycin (100 g/ml), 10% of foetal calfserum and geneticin G418 (400 mg/l) as selecting compound for theLLC-PK1-mdr1a and LLC-PK1-HcPgpA cells. All compounds and medium arefrom Invitrogen, Cergy Pontoise, France. Cells were seeded on 24-wellplates (Sarstedt, Orsay, France) at 2×10⁵ cells/well in G418-free mediumuntil confluence for transport activity and on 96-well plates forviability assay.

Multidrug resistant tumor cells used in the present invention were Humanlymphoma parental CEM and vinblastine-resistant CEM/VLB (Zordan-Nudpo etal., 1993) and parental CEM and multidrug resistant cells DC-3F/ADXselected from spontaneously transformed DC-3F Chinese hamster lungfibroblasts on the basis of their resistance to actinomycin D (Biedlerand Riehm, 1970). Both types of resistant cells overexpressed Pgp.

1.4 Animal Model

Wild-type and the Pgp knock-out mdr1ab^(−/−) mice with a FVB geneticbackground were obtained from Taconic (NY, USA). In rodents, there aretwo Pgps encoded by abc1a and abc1b genes and mdr1ab⁻ mice weredeficient for the two gene products. Mice were housed at INRA'stransgenic rodent facility at 22±2° C. under 12-hour light/dark cycles.Animals sampling was designed to reduce the influence of interferingparameters such as litter specificity (seven to nine different littersfor a ten animals group). Mice received a standard chow diet recommendedfor the breeding and rearing of rodents (Harlan Teklad TRM Rat/MouseDiet, Harlan Teklad, Gannat, France). Water and food were available adlibitum. In vivo studies were conducted in mice under European laws onthe protection of animals and protocols are performed under procedureand principal for good clinical practice.

1.5 Tested Molecules

Ivermectin aglycone obtained according to the synthesis method describedin part 1.1 and purified is used in all the comparative experiments ofthe present invention.

Ivermectin purchased from Sigma is used as inhibition standard in allthe comparative experiments of the present invention.

Valspodar® was kindly provided by Novartis and is used as referenceinhibitor of Pgp.

All the three aforementioned compounds are solubilised in DMSO.

1.6 Transport Tests In Vitro

Cells were cultured with rhodamine 123 (10 μM, purchased from Sigma)with or without valspodar (VSP, 5 μM). Compounds of interest weredissolved in DMSO and diluted in the medium (final DMSOconcentration=0.1%) in a concentration range of 0.1-50 μM. After the 2-hincubation period, the cells were lysed and lysates were stored at −20°C. until analysis. To study the Pgp transport activity, theintracellular accumulation of fluorescent Rho 123 was determined byreading fluorescence in the cell lysates with a spectrofluorimeter(PerkinElmer LS50B, max excitation=507 nm; max emission=529 nm). Proteinconcentration was determined in lysates with BCA kit using bovine serumalbumin as protein standard (Thermo scientific) Results were expressedas fluorescence arbitrary units after normalization to cellular proteincontent per well.

1.7 GABA Receptor Affinity Test

The ability of ivermectin or moxidectin or ivermectin aglycone orivermectin monosaccharide to interact with GABA receptors is assayed byelectrophysiology measurements. Xenopus laevis oocytes are injected with46 nl of RNA solution, with RNA coding for α₁, β₂ and γ₂ subunits of theGABA channel at a ratio of 10:10:50 nM. The injected oocytes areincubated in modified Barth's solution [90 mM NaCl, 3 mM KCl, 0.82 mMMgSO₄, 0.41 mM CaCl₂, 0.34 mM Ca(NO₃)₂, 100 U/ml penicillin, 100 μg/mlstreptomycin and 100 μg/ml kanamycin, 5 mM HEPES pH 7.6] at 18° C. forapproximately 36 h before the measurements to ensure the expression of afunctional receptor.

Electrophysiological experiments are performed by the two-electrodevoltage-clamp method. Measurements were done in ND96 medium containing96 mM NaCl, 2 mM KCl, 1 mM g Cl₂, 1.8 mM CaCl₂ and 5 mM HEPES, pH 7.5,at a holding potential of −80 mV. The control current is evoked by theapplication of 2 μM GABA and the normalized relative potentiation of 2μM GABA-evoked currents by increasing concentration of ivermectin,moxidectin, ivermectin aglycone, or ivermectin monosaccharide isdetermined as:

[(I_(MLs+2 μM GABA)/I_(2 μM GABA alone))/(I_((MLs+2 μM GABA)Max)/I_(2 μM GABA alone))]×100%where I_(2 μM GABA) is the control current evoked by 2 μM GABA,I_(MLs+2 μM GABA) is the current evoked by each drug concentration inco-applications with 2 μM GABA, and I_((MLs+2 μM GABA)Max) is themaximal current evoked by co-applications of drugs and 2 μM GABA. Awashout period of 4 min between each GABA application is introduced,allowing receptors to recover from desensitization. Three differentbatches of oocytes are used to collect data for each analysis. Theperfusion system is cleaned between two experiments by washing with 10%DMSO after application of MLs derivatives to avoid contamination.

1.8 In Vivo Toxicity Test

Toxicity of ivermectin and ivermectin aglycone is measured inPgp-deficient mice. Mdr1ab^(−/−) mice are injected subcutaneously withincreasing doses of ivermectin or ivermectin aglycone formulated inpropylene glycol/formaldehyde (60:40, v/v). Higher injected doses are1.5 mg/kg (1.7 μmol/kg) for ivermectin and 16 mg/kg (27 μmol/kg) forivermectin aglycone, respectively. Toxicity is evaluated during 24 h. Atthe end of the monitoring, plasma is collected, from the orbital sinusvein under methoxyflurane anesthesia and the mice are sacrificed for thebrain collection. Blood is centrifuged at 1500 g for 10 min, and plasmais stored at −20° C. until analysis. The brains is removed, washed insaline solution, and frozen at −20° C. until analysis.

1.9 Ivermectin Resistance Assay in Caenorhabditis elegans

A gravid assay method, based on the development of eggs to gravid adultsover a 96 hr incubation period, was used to determine the resistancewith respect to ivermectin (IVM) in C. elegans. The eggs were collectedthrough rinsing the C. elegans worms resistant to IVM (IVR10). Sixtyeggs were incubated/well, in standard conditions for four days (96hours) in order reach adulthood (gravid) in the presence of drugs asfollowed: ivermectin aglycone (IVM-Agly) alone at 10 ng/ml (11.4 nM);verapamil (VRP) alone at 8 μM; IVM alone: 0, 1, 2, 4, 6, 8, 10, 20 nM;IVM+VRP 8 μM: 0, 1, 2, 4, 6, 8, 10, 20 ng/ml IVM; IVM+IVM-Agly 10 ng/ml:0, 1, 2, 4, 6, 8, 10, 20 ng/ml (0.114-22.8 nM) IVM. Assays wereperformed in triplicates per condition treatment and the experiment wasperformed 3 times.

2. Results

2.1 Ivermectin Aglycone Synthesis

Ivermectin aglycone is obtained from ivermectin by acid hydrolysis,which cuts the chemical bond between macrocycle and disaccharide group.The product obtained after this reaction is a mixture of about 80%ivermectin aglycone and 20% monosaccharide of ivermectin, as showed bystructure profile performed by HPLC (FIGS. 1A and 1B). Ivermectinaglycone obtained by said synthesis method is characterised by ahydroxyl group on carbon C13 of macrocycle (FIG. 1B) and a 3 minutes ofretention time in our chromatographic conditions, shorter than that ofivermectin (5 minutes) or that of monosaccharide derivative (4 minutes).

The obtained product is then analysed by mass spectrometry, whichconfirms the presence of a mass pick at 609.3 which corresponds toionised ivermectin aglycone (FIG. 2), while the mass pick of nativeivermectin aglycone is at 586.8.

2.2 Ivermectin Aglycone Inhibitory Potency for Pgp in Cell Model

Ivermectin aglycone inhibitory potency for transport activity of Pgp isassayed in transfected cells LLCPK1-mdr1a overexpressing murine Pgp(mdr1a). Maximum inhibition has been obtained with Valspodar®, the mostpowerful reference inhibitor of Pgp known in the past. It is confirmedthat ivermectin is an inhibitor of Pgp as powerful as Valspodar® (FIG.3). It is also shown that ivermectin aglycone has a comparable efficacyto inhibit murine Pgp to that of ivermectin, with maximal effect(E_(max)) at about 10 μM (Table 1, FIG. 3).

TABLE 1 Inhibitory effect of ivermectin and ivermectin aglycone in cellsoverexpression murine Pgp Ivermectin Ivermectin aglycone EC₅₀ (μM) 0.51.0 C_(max) (μM) 5.0 10.0 E_(max) (% valspodar ®) 88.0 80.0 EC₅₀:effective concentration for inhibiting 50% of transport of rhodamine 123by murine Pgp. C_(max): concentration to obtain maximum inhibitoryeffect. E_(max): maximum effect compared to maximum effect obtained with5 μM of valspodar.

2.3 Different Inhibitory Potency of Ivermectin Aglycone for Murine Pgpand Nematode Pgp

Inhibitory potency of ivermectin aglycone or ivermectin for murine Pgpor nematode Pgp is respectively measured in cells LLCPK1-mdr1a, whichoverexpress murine Pgp (MDR1), or in cell model developed by R.Prichard, which overexpress nematode Haemonchus contortus Pgp: hc-pgpA.The results show that ivermectin has similar potency to inhibitmammalian Pgp (EC₅₀=0.5 μM) and nematode HcPgpA (EC₅₀=0.6 μM) (Table 2,FIGS. 4A and 4B), while ivermectin aglycone has 5 times higherinhibitory potency for parasite HcPgpA (EC₅₀=0.5 μM) than for mammalianPgp (EC₅₀=2.5 μM). These results clearly indicated that ivermectinaglycone is more potent in inhibiting nematode HcPgpA than mammalianPgp.

TABLE 2 Concentration of half inhibitory effect of ivermectin andivermectin aglycone in cells overexpression Pgp EC50 μM IvermectinIvermectin aglycone Murine Pgp 0.5 2.5 Nematode PgpA 0.6 0.5

2.4 Ability of Ivermectin Aglycone or Ivermectin Monosaccharide to OpenGABA Receptor in Presence of GABA.

of the ability of ivermectin aglycone or ivermectin monosaccharide topotentiate GABA action on GABA receptor, was assayed according to theprotocol described in aforementioned part 1.7, and was compared withivermectin.

The results displayed in table 4 show that ivermectine monosaccharide(IVM Monosaccharide) and ivermectine aglycone (IVM-Agly) are a weakagonist (EC₅₀=122.4 nM for IVM Monosaccharid and EC₅₀=215.1 nM forIVM-Agly) compared to ivermectin (EC₅₀=29 nM) (Table 3, FIG. 5). Thisresult means that ivermectin monosaccharide and ivermectin aglycone havea much weaker neurotoxicity when compared with ivermectin, and apharmaceutical use of ivermectin aglycone or ivermectin monosaccharideis possible.

TABLE 3 Parameters of interaction of IVM and derivatives with GABAreceptors: EC₅₀ is the concentration needed to induce half of themaximal potentiation of GABA effect by MLs or derivatives. MLs EC₅₀ (nM)MOX 5.6 ± 1.5 IVM 29.3 ± 3.4  IVM Monosaccharide 122.4 ± 20.3  IVMAglycone 215.1 ± 12.45

2.5 In Vivo Toxicity of Ivermectin Aglycone

In vivo toxicity text in Pgp-deficient mice confirms that the lethaldose for ivermectin is from 0.6 to 0.8 mol/kg, as what is described bySchinket et al. (Cell (1994) 77, 491-502). On the contrary, ivermectinaglycone does not show any toxicity when it is administered with a dosetill 10 times higher than that of ivermectin (FIG. 6). This resultconfirms that ivermectin aglycone has a much weaker in vivo toxicitycompared to ivermectin and a pharmaceutical use of ivermectin aglyconeis possible.

2.6 Reversal of Multidrug Resistance by Ivermectin Aglycone in MultidrugResistant Tumor Cells

CEM/VLB cells and DC-3F/ADX cells described in aforementioned part 1.3were plated into 96 well plates and allowed to grow for 24 h. They werethen incubated 4 days with vinblastine (concentration range 0-1 μM) withor without IVM at 2.5 μM or ivermectin aglycone (IVM-Agly) at 2.5 and at5 μM (FIG. 7); or 2 days with actinomycin D with actinomycin(concentration range 0.01-10 μM) with or without ivermectin (IVM) orivermectin aglycone (IVM-Agly) at 5 μM (FIG. 8). Cytotoxicity wasdetermined using the MTT test. IC₅₀ values were graphically determinedand they represent the concentration needed for half cell survival. Foldreversal of multidrug resistance called reversion factors were the ratioof IC₅₀ for toxic drug alone/IC₅₀ for toxic drug in the presence ofIVM-Agly.

IVM-Agly was able to reverse drug resistance in tumor cellsoverexpressing Pgp. CEM/VBL are highly resistant to VBL and cells werefully viable in 1 μM vinblastine while the parental cells are highlysensitive to VBL at concentrations below 0.001 μM. Co-incubation of VBLwith IVM at 2.5 μM, or IVM-Agly at 5 μM provoke a clear left-shift ofthe viability cell curve (FIG. 7) demonstrating that cells aresensitized to VBL in presence of the tested compounds. In the presenceof IVM at 2.5 μM the VBL IC₅₀ was 0.2 μM and in presence of IVM-Agly at2.5 and 5 μM, the IC₅₀ values were 1 and 0.2 μM, reflecting thatIVM-Agly's has similar inhibitory potency compared to that of IVM (Table1). In addition, DC-3F/ADX viability was not altered by 1 μM actinomycinD while when combined with IVM or IVM-Agly at 5 μM actinomycin D becametoxic (FIG. 8).

The results of FIG. 7, FIG. 8 and table 4 showed that the ability ofivermectin aglycone to reverse vinblastine or actinomycin D-resistancein tumor cells overexpression Pgp was of the same order of potency asivermectin, which is potent inhibitor of MDR transporters.

TABLE 4 Comparison of IC50 and resistance factor (RF) for IVM and IVMAgly in multidrug-resistant cells IC50 (μM) RF CEM/VBL VBL Nd VBL + IVM2.5 μM 0.2 VBL + IVM-Agly 2.5 μM 1.0 VBL + IVM-Agly 5 μM 0.2 DC-3F/ADXActD 5.0 ActD + IVM 5 μM 0.11 45 ActD + IVM-Agly 5 μM 0.08 62 Nd: notdetermined

2.7 Reversal of Anthelmintic Resistance by Ivermectin Aglycone in C.elegans Resistant to Ivermectin

The reversal action of ivermectin aglycone (IVM-Agly) was studied on thenematode Caenorhabditis elegans resistant to ivermectin (IVR10). Thisstrain has been previously selected under IVM pressure and it was shownto overexpressed P-gp homologue genes (James and Davey, 2009). Wemeasured the ability of IVM-Agly to restore the development from eggs toadults which has been delayed by the ivermectin effect on the IVR10strain, and compared its effect to that of the verapamil (VRP) reversaleffect.

The resistance with respect to ivermectin in C. elegans is measuredaccording to the protocol described in aforementioned part 1.9.

IVM blocked the development of C. elegans IVR10 eggs at a concentrationaveraging 10 nM confirming that this strain is resistant to IVM. TheIC₅₀ for IVM was 6.8±0.2 ng/ml (7.8±0.2 nM). verapamil, a knownPgp-reversing agent, at 8 μM had no effects on the development of the C.elegans when alone, and was able to restore the development of wormsstopped in the presence of IVM. The curve of IVM efficacy was thusshifted to the left with the IC₅₀ of IVM reduced to 3.2±0.5 ng/ml(3.6±0.6 nM) when compared to IVM alone (FIG. 9, Table 5). IVM-Agly at10 ng/ml was also able to significantly decrease the EC₅₀ of IVM to4.5±0.3 ng/ml (5.1 nM, Table 5), and IVM-Agly alone at 10 ng/ml had noeffects on the development of the C. elegans suggesting that IVM-Aglyalso reverse a Pgp-mediated drug resistance.

The lower EC₅₀ for ivermectin efficacy in IVM resistant C. elegansdetermined in presence of IVM-Agly testifies that IVM-Agly is able topartly reverse IVM resistance. Based on the fact that verapamil arewell-known inhibitors of Pgp, their effects comparable to the oneproduced by IVM-Agly suggest that the IVM-Agly reversion also occursthrough inhibition of Pgp-like transporters.

TABLE 5 Comparison of IC₅₀ and resistance factor (RF) for the referencereversal agent valspodar and verapamil and IVM-Agly inivermectin-resistant C. elegans IC₅₀ (nM) RF IVM alone 7.8 ± 0.2 IVM +verapamil (4 μM) 3.6 ± 0.6 2.1 IVM + IVM-agly (11.4 nM) 5.1 ± 0.3 1.5

What is claimed:
 1. A pharmaceutical composition comprising (a) acompound of formula I

wherein (i) R₁ is selected from the group consisting of —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, and cyclohexyl, (ii) X represents —CH₂—CH₂—, or —CH═CH—,(iii) R₂ is

(iv)

R₃ is OH or NOH, and

represents a single bond when R₃ is OH, or a double bond when R₃ is NOH;(b) a second active ingredient selected from the group consisting of amacrocyclic lactone antiparasitic agent other than ivermectin, anantitumoral agent, an antiviral agent, an anti-epileptic agent, anantibacterial agent, and an antifungal agent, wherein the second activeingredient is a compound other than a compound of formula I; and (c) apharmaceutically acceptable carrier, wherein pharmaceutical compositionis formulated for oral administration, subcutaneous injection,intravenous injection, or intra-tissue injection, and wherein, thecompound of formula I is present in the composition in an amount thatreduces resistance of target cells, organisms, or parasites to thesecond active ingredient that occurs in the absence of the compound offormula I.
 2. The medicament pharmaceutical composition of claim 1,wherein the compound is a monosaccharide of ivermectin of formula I(b):

wherein (i) R₁ is selected from the group consisting of —CH(CH₃)₂ and—CH(CH₃)CH₂CH₃, (ii) X represents —CH₂—CH₂—, and (iii)

R₃ is —OH.
 3. The pharmaceutical composition of claim 1, wherein thecompound is a compound of formula I(c):

wherein (i) R₁ is selected from the group consisting of —CH(CH₃)₂ and—CH(CH_(J))CH₂CH₃ , (ii) X represents —CH═CH—, and (iii)

R₃ is —OH.
 4. The pharmaceutical composition of claim 1, wherein thecompound is a compound of formula I(d):

wherein (i) R₁ is cyclohexyl, (ii) X represents —CH═CH—, and (iii)

R₃ is —OH.
 5. The pharmaceutical composition of claim 1, wherein thecompound is a selamectin or selamectin aglycone of formula I(e):

wherein (i) R₁ is cyclohexyl, (ii) X represents —CH₂—CH₂—, and (iii)

R₃ is ═NOH.
 6. The pharmaceutical composition of claim 1 wherein thesecond active ingredient is selected from the group consisting ofavermectins and milbemycins.
 7. The pharmaceutical composition of claim1 wherein the second active ingredient is an antiparasitic agent.
 8. Thepharmaceutical composition of claim 7 wherein the second activeingredient is chosen from an antiplasmodium or antileshmanial agent. 9.The pharmaceutical composition of claim 1 wherein the second activeingredient is an antitumoral agent.
 10. The pharmaceutical compositionof claim 9 wherein the second active ingredient is an anthracycline, ataxane, an alkaloid, or an epipodophyllotoxin.
 11. The pharmaceuticalcomposition of claim 1 wherein the second active ingredient is anantiviral agent.
 12. The pharmaceutical composition of claim 1 whereinthe second active ingredient is a protease inhibitor.
 13. Thepharmaceutical composition according to claim 1, wherein saidpharmaceutical composition consists of the compound of formula I, thesecond active ingredient, and the pharmaceutically acceptable carrier.14. A method of treatment of infections, cancers that are resistant tochemotherapies other than those involving use of the medicament of claim1, or epilepsy which method comprises administering to a subject in needof such treatment a pharmaceutical composition comprising (a) a compoundof formula I

wherein (i) R₁ is selected from the group consisting of —CH(CH₃)₂,—CH(CH₃)CH₂CH₃, and cyclohexyl, (ii) X represents —CH₂—CH₂—, or —CH═CH—,(iii) R₂ is

(iv)

R₃ is OH or NOH, and

represents a single bond when R₃ is OH, or a double bond when R₃ is NOH;(b) a second active ingredient selected from the group consisting of amacrocyclic lactone antiparasitic agent other than ivermectin, anantitumoral agent, an antiviral agent, an anti-epileptic agent, anantibacterial agent, and an antifungal agent, wherein the second activeingredient is a compound other than a compound of formula I; and (c) apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for oral administration, subcutaneousinjection, intravenous injection, or intra-tissue injection, and whereinthe pharmaceutical composition reduces resistance of target cells,organisms, or parasites as compared to administration of the secondactive ingredient without the compound of formula I.
 15. The method ofclaim 14 for the treatment of a parasitic infection wherein the secondactive ingredient is an antiparasitic agent.
 16. The method of claim 14for the treatment of a viral infection, a bacterial infection or afungal infection wherein the second active ingredient is an antiviralagent, antibacterial agent or an antifungal agent, respectively.
 17. Themethod of claim 14 for the treatment of chemotherapy resistant cancerswherein the second active ingredient is an antitumoral agent.
 18. Themethod of claim 14 for the treatment of epilepsy wherein the secondactive ingredient is an anti-epileptic agent.
 19. The pharmaceuticalcomposition of claim 1, wherein the second active ingredient is anantiparasitic agent selected from abamectin, doramectin, eprinomectin,or a milbemycin.
 20. The pharmaceutical composition of claim 1, whereinthe reduction of resistance to the second active agent is determined bya reduction of the IC₅₀ in cells overexpressing P-glycoprotein when thecells are contacted with the pharmaceutical composition, as compared tothe IC₅₀ when the cells are contacted with the second active ingredientwithout the compound of formula I.